Heat-stable dry powder pharmaceutical compositions and methods

ABSTRACT

Disclosed herein are heat-stable dry powders which include peptides or protein such as oxytocin for use as a pharmaceutical composition. The composition is highly stable at increased temperatures and relatively high humid environments, and are intended for storage at room temperature with an improved shelf-life. In particular, the dry powders are intended for inhalation, however, other routes of administration can be used when reconstituted in solution.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/905,236, filed Jan. 14, 2016, which is a 371 of PCT/US2014/047304,filed Jul. 18, 2014, which claims benefit under 35 U.S.C. § 119(e) fromU.S. Provisional Patent Application Ser. No. 61/847,981, filed Jul. 18,2013, the content of which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

Disclosed herein are heat-stable dry powder compositions and methods fordelivering biodegradable substances, including peptides and proteins,and systems and methods for delivering the dry powders. In particular,the dry powders are preferably intended for pulmonary delivery byinhalation to treat certain disorders and/or diseases, includingpost-partum hemorrhaging.

BACKGROUND

Delivery of drugs has been a major problem for many years, particularlywhen the compound to be delivered is unstable under the conditionsencountered in the gastro-intestinal tract when administered orally to asubject, prior to reaching its targeted location. For example, it ispreferable in many cases to administer drugs orally especially in termsof ease of administration, patient compliance, and decreased cost.However, many compounds are ineffective or exhibit low or variablepotency when they are administered orally. Presumably this is becausethe drugs are unstable under conditions in the digestive tract orbecause they are inefficiently absorbed. For biologic products, inparticular peptides and proteins, the acidic environment in the stomachis detrimental to maintain function as most proteins are degradedreadily.

Isolated biological substances, including, certain proteins and peptidescan readily and completely lose functional activity, for example, bytaking them out of −20° C. storage once. Other isolated proteins andpeptides undergo significant degradation when stored at 4° C., withoutthe addition of protease inhibitors. Most mammalian proteins andpeptides degrade at a temperature greater than 43° C. It has been wellestablished that at 55° C., most proteins undergo complete denaturationin about 1-2 hours. In some cases, complete denaturation anddestabilization of an isolated protein also occurs at room temperature.

Due to the problems associated with oral drug delivery of drugs and inparticular, biologically-derived products, drug delivery to the lungshas been explored. For example, drugs delivered to the lungs aredesigned to have an effect on the tissue of the lungs, for example,vasodilators, surfactants, chemotherapeutic agents or vaccines for fluor other respiratory illnesses. Drug formulations for treating pulmonarydiseases such as asthma are available by several methods, including,using nebulizers such as treatment with PULMOZYME®, using metered-doseinhalers such as SYMBICORT®, and dry powder inhalers such as ADVAIRDISKUS®, PULMICORT FLEXAHER®. Other drugs, including nucleotide drugs,have been delivered to the lungs because they represent a tissueparticularly appropriate for treatment, for example, for genetic therapyin cystic fibrosis, where retroviral vectors expressing an effectiveadenosine deaminase are administered to the lungs.

Currently, formulations for treating systemic disease using biologicproducts are available primarily through injectable compositions. Drypowder compositions for pulmonary inhalation and systemic delivery ofinsulin have been used including EXUBERA®, and AFREZZA® in clinicaltrials. There is the desire, however, to improve the shelf-life at roomtemperature for dry powder compositions, especially those comprising abiologic molecule, including peptides and nucleic acids, to furtherprolonged their life, facilitate their storage and delivery prior topatient use, particularly if refrigeration is not available.

For example, according to the World Health Organization, 800 women dieevery day from pregnancy or childbirth-related complications. Among themajor causes of death is severe bleeding (post-partum hemorrhage) thatcan be prevented by the use of a peptide hormone, oxytocin, a biologicmolecule. Commercially available oxytocin compositions are provided asliquid formulations under the trade names PITOCIN® and SYNTOCINON® or asgeneric oxytocin; the peptide in solution degrades readily at ambienttemperature, requires storage below 25° C. prior to use, and isadministered only by injection. The preparations of injectableformulations and special storage needed create challenges, whichprohibit their use in subtropical and tropical climates where there is agreat need, and refrigeration and sterilization are not always readilyavailable.

Accordingly, there is room for improvement in the development ofpharmaceutical formulations comprising biologic molecules in particularfor pulmonary delivery in the treatment of disease.

SUMMARY

The present disclosure provides dry powder compositions for inhalationwhich are stable at room temperature or higher temperatures forprolonged periods of time without substantially losing their biologicalactivity. In one embodiment, a pharmaceutical formulation is providedcomprising a dry powder for inhalation comprising a biologic molecule,wherein the biologic molecule comprises a peptide or a protein forsystemic delivery using a dry powder inhalation system comprising aninhaler that can be used with a unit dose cartridge or capsule formultiple use, a single use inhaler with an integrally built-in containerfor single use, or a multidose inhaler provided with a plurality ofdoses integrally configured with the inhaler.

In one embodiment, a heat-stable pharmaceutical formulation is providedcomprising, a dry powder comprising a protein or a peptide and one ormore pharmaceutically acceptable carriers and/or excipients, whichformulations are stable at high temperatures and high humidity. In oneembodiment, the pharmaceutical formulation is stable for a long periodof time at temperatures, for example, temperatures greater than 20° C.,greater than 25° C., greater than 30° C., or greater than 35° C.; andrelative humid environments such as environments having a relativehumidity greater than 5%, greater than 10%, greater than 30%, greaterthan 50%, greater than 60%, or greater than 70%; wherein thepharmaceutically acceptable carriers and/or excipients include, forexample, buffers, salts, polymers, diketopiperazines and/or saltsthereof, and the like. In one embodiment, the dry powder compositionscan optionally include surfactants such as polysorbates, for example,polysorbate 80 and Tween.

In a certain embodiments, the formulation comprises a dry powdercomprising a peptide, including, for example, oxytocin, an oxytocinderivative or an analog thereof such as carbotecin; a buffer, and amonovalent or divalent cationic salt, and optionally a sugar and/or anamino acid. In a particular embodiment, the formulation comprises a drypowder comprising oxytocin, an oxytocin derivative, or an oxytocinanalog; a buffer and/or a divalent cation or monovalent cation providedby a salt, including, zinc citrate, zinc acetate, disodium tartrate,mono-sodium tartrate, sodium citrate, disodium citrate, trisodiumcitrate, zinc chloride, calcium chloride, magnesium chloride, sodiumhydroxide, and the like. In one embodiment, the formulation furthercomprises one or more amino acids, including leucine, isoleucine,trileucine, cystine, arginine, lysine, methionine, and/or histidine. Inan embodiment, the monovalent cation in the formulation can includesodium, potassium and lithium. In an alternate embodiment, theformulation may be provided with citric acid.

In a specific embodiment, a dry powder composition is providedcomprising oxytocin, sodium citrate, including, monovalent, divalent ortrivalent form, in an amount less than 40% (w/w), less than 30% (w/w),less than 20% (w/w), or less than 10% (w/w), and zinc chloride or zinccitrate in an amount less than 35% (w/w), less than 20% (w/w), or lessthan 10% (w/w) in the composition. In a particular embodiment, the zincchloride is used in an amount ranging from about 1% to about 7% (w/w) ofthe composition. In an alternative embodiment, the zinc citrate is usedin an amount ranging from about 9% to about 35% (w/w) of thecomposition.

In a specific embodiment, a dry powder composition is providedcomprising oxytocin, sodium tartrate, including, monovalent, or divalentform, in an amount less than 40% (w/w), less than 30% (w/w), less than20% (w/w), or less than 10% (w/w), and zinc chloride or zinc citrate inan amount less than 35% (w/w), less than 20% (w/w), or less than 10%(w/w) in the composition. In a particular embodiment, the zinc chlorideis used in an amount ranging from about 1% to about 7% (w/w) of thecomposition. In an alternative embodiment, the zinc tartrate is used inan amount ranging from about 9% to about 35% (w/w) of the composition.

In one embodiment, the dry powder composition comprises citrate salts inan amount ranging from 100 to 20 equivalents per mole of oxytocin, anoxytocin analog or derivative thereof; and the amount of zinc salts canrange from 50 to 5 equivalents per mole of oxytocin in the composition.In some embodiments, concentrated sodium citrate buffers were used asthe source of citrate; wherein the citrate buffers had a concentrationup to 0.1 M or 0.75 M and range in pH values of 4.0 to 6.5.

In one embodiment, the dry powder composition comprises oxytocin or ananalog or derivative thereof; zinc and citrate, wherein the oxytocin,analog or derivative thereof is in an amount up to 200 IU in a singleinhalable dose. In some embodiments, the dry powder compositioncomprises 150 IU, 100 IU, 50 IU, 40 IU, 20 IU, 10 IU, 5 IU, 1 IU, 0.05IU, or 0.005 IU of oxytocin, an analog or a derivative thereof in asingle inhalable dose.

A method of making a dry powder formulation comprising mixing orhomogenizing a solution comprising a peptide or protein or analogthereof, wherein the solution comprises citrate salts in an amountranging from 100 to 20 equivalents per mole of the peptide or protein;and an amount of zinc salts can range from 50 to 5 equivalents per moleof the peptide or protein or analog thereof in the composition. In someembodiments, concentrated sodium citrate buffers were used as the sourceof citrate; and spray-drying a solution in a nitrogen gas chamber,comprising a peptide, protein, fragments thereof and/or analogs thereof,wherein the dry powder formulation comprises a mixture of the peptide,protein, fragments thereof and/or analogs thereof; a citrate or tartrateand a cationic salt at a pH ranging from pH 4.5 to pH 6.5, and whereinthe cationic salt is a divalent cationic salt.

Embodiments include a method for treating post-partum hemorrhagingcomprising administering to a subject in need of treatment a dry powderformulation by inhalation, the composition comprising oxytocin, ananalog thereof or derivative thereof; a citrate or tartrate and a sourceof a cation, including, zinc within 24 hours post-partum. In oneembodiment, the treatment comprises administrating one or more doses ofthe dry powder formulation described herewith immediately uponchildbirth.

In an alternate embodiment, a method of preventing post-partumhemorrhage comprising administering to a subject susceptible ofpost-partum hemorrhage a dry powder formulation comprising oxytocin, ananalog thereof or derivative thereof; a citrate or tartrate, and asource of a cation, including, zinc within 24 hours or immediately afterchildbirth.

In other embodiments described herewith, there are disclosed methods formaking heat-stable and humidity-stable formulations and methods forusing the formulations in the treatment of diseases and/or disordersincluding, for example, post-partum hemorrhaging, autism, social anxietydisorders; mood disorders, and other hormone-related diseases, inembodiments using an inhalation system. In an exemplary embodiment, theinhalation system is a high resistance inhaler for single dose usage.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the examplesdisclosed herein. The disclosure may be better understood by referenceto one or more of these drawings in combination with the detaileddescription of specific embodiments presented herein.

FIGS. 1A and 1B are scanning electron micrographs of an amorphous drypowder formulation embodiment comprising 1% oxytocin, 87% trehalose; 10%isoleucine and 10% polyvinylpirrolidone (PVP) at low (1A) and highmagnification (1B).

FIG. 2A is a scanning electron micrograph of a control powder similar toFIG. 1B at high magnification. FIGS. 2B, 2C and 2D are scanning electronmicrographs of an amorphous dry powder formulation embodimentscomprising 1% oxytocin; citrate and a zinc salt at high magnificationand containing differing amounts of divalent zinc salts and citratesalts.

FIG. 3 provides a graphic representation of data obtained from stabilitystudies data for dry powder composition embodiments comprising 1%oxytocin incubated at 40° C. and 75% relative humidity for a period ofapproximately 11 months compared to a control.

FIG. 4 provides a graphic representation of data obtained from X-raydiffraction studies of dry powders showing amorphous content of thepowders by their characteristic scan patterns.

FIG. 5 is a graphic representation of dry powder samples from thestability studies wherein the samples contained divalent zinc salt andcitrate salts at various concentrations.

DETAILED DESCRIPTION

Drug delivery to the lungs offers many advantages. It is difficult todeliver drugs into the lungs due to problems in transporting the drugspast natural physical barriers in a uniform volume and weight of thedrug and the drug physical and chemical characteristics. Disclosedherein are heat-stable formulations comprising, a buffer, including,citrate, and a monovalent, or divalent cation, and one or morepharmaceutically acceptable carriers and/or excipients. Embodimentsdisclosed herein show that the dry powder formulations are stable athigh heat and humidity and thus they facilitate and overcome the storageand refrigeration challenges posed by prior art formulations. A methodof making the dry powder composition for extended storage attemperatures greater than 20° C. and humid environments if alsoprovided.

As used herein, the term “microparticle” refers to a particle with adiameter of about 0.5 to about 1000 μm, irrespective of the preciseexterior or interior structure. Microparticles having a diameter ofbetween about 0.5 and about 10 microns can reach the lungs, successfullypassing most of the natural barriers. A diameter of less than about 10microns is required to navigate the turn of the throat and a diameter ofabout 0.5 microns or greater is required to avoid being exhaled. Toreach the deep lung (or alveolar region) where most efficient absorptionis believed to occur, it is preferred to maximize the proportion ofparticles contained in the “respirable fraction” (RF), generallyaccepted to be those particles with an aerodynamic diameter of about 0.5to about 6 microns, though some references use somewhat differentranges, as measured using standard techniques, for example, with anAnderson Cascade Impactor. Other impactors can be used to measureaerodynamic particle size such as the NEXT GENERATION IMPACTOR™ (NGI™,MSP Corporation), for which the respirable fraction is defined bysimilar aerodynamic size, for example <6.4 μm. In some embodiments, alaser diffraction apparatus is used to determine particle size, forexample, the laser diffraction apparatus disclosed in U.S. patentapplication Ser. No. 12/727,179, filed on Mar. 18, 2010, which isincorporated herein in its entirety for its relevant teachings relatedto laser diffraction, wherein the volumetric median geometric diameter(VMGD) of the particles is measured to assess performance of theinhalation system. For example, in various embodiments cartridgeemptying of ≥80%, 85%, or 90% and a VMGD of the emitted particles of≤12.5 μm, ≤7.0 μm, or ≤4.8 μm can indicate progressively betteraerodynamic performance.

As used herein, the term “about” is used to indicate that a valueincludes the standard deviation of the measurement for the device ormethod being employed to determine the value.

Respirable fraction on fill (RF/fill) represents the % of powder in adose that is emitted from an inhaler upon discharge of the powdercontent filled for use as the dose, and that is suitable forrespiration, i.e., the percent of particles from the filled dose thatare emitted with sizes suitable for pulmonary delivery, which is ameasure of microparticle aerodynamic performance. As described herein, aRF/fill value of 40% or greater than 40% reflects acceptable aerodynamicperformance characteristics. In certain embodiments disclosed herein,the respirable fraction on fill can be greater than 50%. In an exemplaryembodiment, a respirable fraction on fill can be up to about 80%,wherein about 80% of the fill is emitted with particle sizes <5.8 μm asmeasured using standard techniques.

As used herein, the term “dry powder” refers to a fine particulatecomposition that is not suspended or dissolved in a propellant, or otherliquid. It is not meant to necessarily imply a complete absence of allwater molecules.

As used herein, “amorphous powder” refers to dry powders lacking adefinite repeating form, shape, or structure, including allnon-crystalline powders.

In one embodiment, the dry powder is a relatively cohesive powder whichrequires optimal deagglomeration condition. In one embodiment, theinhalation system provides a re-useable, miniature breath-poweredinhaler in combination with single-use cartridges containing pre-metereddoses of a dry powder formulation.

As used herein the term “a unit dose inhaler” refers to an inhaler thatis adapted to receive or comprises a single container comprising a drypowder formulation and delivers a single dose of a dry powderformulation by inhalation from the container to a user. In someinstances multiple unit doses will be required to provide a user with aspecified dosage. In one embodiment, the inhaler is a dry powderinhaler, which can be disposable for single use, or reusable formultiple uses with a single unit dose container.

As used herein the term “a multiple dose inhaler” refers to an inhalerhaving a plurality of containers, each container comprising apre-metered dose of a dry powder medicament and the inhaler delivers asingle dose of a medicament powder by inhalation at any one time.

As used herein a “container” is an enclosure configured to hold orcontain a dry powder formulation, a powder containing enclosure, and canbe a structure with or without a lid. This container can be providedseparately from the inhaler or can be structurally integrated within theinhaler (e.g. non-removable). Further, the container can be filled witha dry powder. A cartridge can also include a container.

As used herein a “powder mass” refers to an agglomeration of powderparticles or agglomerate having irregular geometries such as width,diameter, and length.

As used herein, the term “microparticle” refers to a particle with adiameter of about 0.5 to about 1000 μm, irrespective of the preciseexterior or interior structure. However four pulmonary deliverymicroparticles that are less than 10 μm are generally desired,especially those with mean particles sizes of less than about 5.8 μm indiameter.

In an exemplary embodiment, a dry powder formulation is provided,comprising, a peptide or a protein, wherein the peptide or protein issensitive to degradation by heat. In a particular embodiment, the drypowder formulation comprises a peptide including, oxytocin, an oxytocinderivative, or an oxytocin analog; a citrate, including; sodium citrateand zinc citrate; a divalent salt; including zinc chloride; calciumchloride and magnesium chloride; and one or more pharmaceuticallyacceptable carriers selected from sugars, for example, saccharides,disaccharides; oligosaccharides; an amino acid; wherein the sugar is,for example, trehalose, mannose, mannitol or sorbitol, and the carrieris polyethylene glycol, polyvinylpyrrolidone, or a diketopiperazinecapable of forming microparticles, including, fumaryl diketopiperazine,succinyl diketopiperazine, maleyl diketopiperazine, malonyldiketopiperazine and oxalyl diketopiperazine, or the disodium ormagnesium salt thereof, and derivatives thereof.

In another embodiment, the formulation comprises a peptide, including,growth hormone, calcitonin, glucagon, parathyroid hormone, parathyroidhormone (1-34), glucagon-like peptide-1, interferon, interleukin,erythropoietin, luteinizing hormone-releasing hormone, somatostatin,vasopressin, enkephalin, adrenocorticotropic hormone, growthhormone-releasing hormone, growth factors, including, granulocyte colonyformation-stimulating factor; thyroid stimulating hormone,thyroid-stimulating hormone-releasing hormone, antinociceptive peptides,angiotensin, prolactin, luteinizing hormone, rennin, gastric inhibitorypolypeptide (GIP), and C-peptide.

In another embodiment, the formulation comprises a peptide, wherein thepeptide is oxytocin, insulin, growth hormone, calcitonin, glucagon,parathyroid hormone, glucagon-like peptide-1, glucagon like-peptide-2,parathyroid hormone (1-34), or parathyroid hormone releasing hormone,oxyntomodulin, peptide YY, leptin, deoxyribonuclease, ribonuclease, andfollicle stimulating hormone.

In one embodiment, the formulation comprises one or more peptides, oneor more amino acid, wherein the amino acid is isoleucine, leucine,trileucine, cystine, cysteine, glycine, lysine, arginine, histidine, ormethionine; and one or more sugars, including, lactose, mannitol,mannose, sorbitol, trehalose, and the like. In this and otherembodiments, the carrier can be polyethylene glycol,polyvinylpyrrolidone, or a saccharide, an oligosaccharide, or apolysaccharides, including lactose, trehalose, mannose, mannitol, orsorbitol; zinc citrate and zinc chloride; wherein the formulation ismade by a spray-drying process wherein the peptide is in a bufferedsolution having a pH ranging from about pH 3.5 to about pH 7; or pH 4.5to pH 6.5.

In a particular embodiment, the formulation comprises oxytocin inconcentration from about 0.005 IU to about 40 IU, from 1 IU to about 15IU; or from about 5 IU to about 20 IU. In one embodiment, oxytocin isadministered to a patient to prevent post-partum hemorrhaging a fewminutes after giving birth in a formulation comprising oxytocin in anamount ranging from 5 to about 40 IU in a single inhalation. In thisembodiment, the content of oxytocin that can be provided in theformulation ranges from about 0.1% (w/w) to about 50% (w/w), from about0.5% (w/w) to about 40% (w/w); from about 0.5% (w/w) to about 20% (w/w);or from about 1% (w/w) to about 10% (w/w). In certain embodiments, theamount of oxytocin can be greater than 40 IU depending in the need ofthe subject to be treated.

In one embodiment, there is provided a method for the effective deliveryof a formulation to the blood stream of a subject, comprising providingto a subject in need of treatment an inhalation system comprising aninhaler including a cartridge containing a formulation comprising a drypowder formulation comprising a peptide including, oxytocin, a citratebuffer or tartrate buffer and a divalent cation salt, wherein thedivalent cation is zinc. In this and other embodiments, the inhalationsystem delivers a powder plume comprising particles having a volumetricmedian geometric diameter (VMGD) less than 8 μm. In an exampleembodiment, the VMGD of the microparticles can range from about 4 μm to6 μm. In an example embodiment, the VMGD of the powder particles can befrom 3 μm to about 6 μm in a single inhalation of the formulation offill mass ranging between 1 mg and 10 mg of dry powder. In this andother embodiments, the inhalation system delivers greater than 40%; orgreater than 60% of the dry powder formulation from the cartridge.

In a further embodiment, the formulation is an amorphous dry powdercomprising microparticles of disodium furmaryl diketopiperazinecomprising oxytocin, a citrate buffer; zinc chloride, an amino acid,such as leucine, isoleucine, trileucine or cystine and mannitol ortrehalose, or a combination thereof.

In an embodiment, the formulation comprises an amorphous dry powdercomprising a peptide, including, a heat-sensitive peptide, includingoxytocin; wherein the dry powder is formed by mixing oxytocin in asolution containing a citrate or acetate buffer at an adjusted pHranging from 4.5 to 6.5 and adding a divalent cationic salt, includingzinc chloride and optionally a sugar such as trehalose or mannitol priorto drying.

In a particular embodiment, the formulation comprises an amorphous drypowder comprising oxytocin; wherein the dry powder is formed by mixingoxytocin in a solution containing citrate salts and/or citric acid andadding a divalent cationic salt, including, zinc chloride and optionallya sugar such as trehalose or mannitol and optionally, one or morecarriers.

Further embodiments concern drug delivery systems comprising an inhaler,a unit dose dry powder medicament container, and a dry powder comprisinga heat-sensitive peptide as disclosed herein and zinc citrate.

One embodiment discloses a formulation comprising oxytocin, a derivativethereof, or an analog thereof, wherein the formulation further comprisesdiketopiperazine microparticles, including, microparticles of fumaryldiketopiperazine having a specific surface area (SSA) of less than about67 m²/g. Another embodiment includes diketopiperazine microparticles inwhich the specific surface area is from about 35 to about 67 m²/g,within a 95% confidence limit. Another embodiment includesdiketopiperazine microparticles in which the specific surface area isfrom about 35 to about 62 m²/g. Another embodiment includesdiketopiperazine microparticles in which the specific surface area isfrom about 40 to about 62 m²/g.

In alternative embodiments, the FDKP microparticles comprise a drug oractive agent. In various embodiments of the FDKP microparticles, thedrug can be, for example, a peptide, including, oxytocin, insulin,glucagon-like peptide-1 (GLP-1), glucagon, exendin, parathyroid hormone,calcitonin, oxyntomodulin, derivatives and/or analogs thereof, and thelike. In another embodiment of the FDKP microparticles, the peptidecontent can vary depending on downstream processing conditions. In aparticular example, the FDKP microparticles can be prepared to have adrug/peptide content that can vary depending on the dose to be targetedor delivered. For example, wherein the drug is insulin, the insulincomponent can be from about 3 U/mg to about 6 U/mg in the powderformulation comprising the microparticles and the zinc salt and citratecan be added to solution prior to forming the particles. In certainembodiments, the drug is adsorbed to the surfaces of pre-formedmicroparticles.

Further embodiments concern drug delivery systems comprising acombination of an inhaler, a unit dose dry powder medicament container,for example, a cartridge, and comprising the dry powder formulationsdisclosed herein and an active agent. In one embodiment, the deliverysystem for use with the dry powders includes an inhalation systemcomprising a high resistance inhaler having air conduits which impart ahigh resistance to airflow through the conduits for deagglomerating anddispensing the powder. In one embodiment, the inhalation system has aresistance value of, for example, approximately 0.065 to about 0.200(√kPa)/liter per minute. In certain embodiments, the dry powders can bedelivered effectively by inhalation with an inhalation system whereinthe peak inhalation pressure differential can range from about 2 toabout 20 kPa, which can produce resultant peak flow rates of aboutbetween 7 and 70 liters per minute. In certain embodiments, theinhalation system are configured to provide a single dose by dischargingpowder from the inhaler as a continuous flow, or as one or more pulsesof powder delivered to a patient. In some embodiments disclosedherewith, the dry powder inhaler system comprises a predetermined massflow balance within the inhaler. For example, a flow balance ofapproximately 10% to 70% of the total flow exiting the inhaler and intothe patient is delivered by one or more dispensing ports, which airflowpasses through the area containing the powder formulation, and whereinapproximately 30% to 90% air flow is generated from other conduits ofthe inhaler. Moreover, bypass flow, or flow not entering and exiting thearea of powder containment such as through a cartridge, can recombinewith the flow exiting the powder dispensing port within the inhaler todilute, accelerate and ultimately deagglomerate the fluidized powderprior to exiting the mouthpiece. In one embodiment, flow rates rangingfrom about 7 to 70 liters per minute result in greater than 75% of thecontainer or the cartridge contents dispensed in fill masses between 1mg and 50 mg; or 1 mg to 30 mg. In certain embodiments, an inhalationsystem as described above can emit a respirable fraction/fill of apowder dose at percentages greater than 40% in a single inhalation,greater than 50%, greater than 60%, or greater than 70%.

In particular embodiments, an inhalation system is provided comprising adry powder inhaler, and a dry powder formulation. In some aspects ofthis embodiment of the inhalation system, the dry powder formulation isprovided in a unit dose cartridge. Alternatively, the dry powderformulation can be preloaded in the inhaler. In this embodiment, thestructural configuration of the inhalation system allows thedeagglomeration mechanism of the inhaler to produce respirable fractionsgreater than 50%; that is, more than half of the powder contained in theinhaler (cartridge) is emitted as particles of less than 5.8 μm. Theinhalers can discharge greater than 85% of a powder medicament containedwithin a container during dosing. In certain embodiments, the inhalerscan discharge greater than 85% of a powder medicament contained in asingle inhalation. In one embodiment, the inhalers can discharge greaterthat 90% of the cartridge contents or container contents in less than 3seconds at pressure differentials between 2 and 5 kPa with fill massesranging up to 30 mg.

Another embodiment disclosed herein includes a method of makingmicroparticles suitable for pulmonary administration as a dry powderformulation comprising, a carrier particle, including, diketopiperazinemicroparticles. In this and other embodiments, the dry powderformulation is obtained by spray-drying a solution containing a peptide,wherein the one or more excipients is dissolved in an aqueous solutioncomprising the zinc salt and citrate and mixed, followed by adding theamount of the peptide with mixing to form a feed solution; atomizing theflow of solution into a drying nitrogen gas flow at an inlet temperatureof about 120° C. to 150° C. and an outlet temperature of about 60° C. to65° C., or 50° C. to 75° C., or 40° C. to 85° C., or the like.

In some embodiments, the method of making diketopiperazinemicroparticles having the specific surface area of less than about 67m²/g, and/or a trans isomer ratio of about 45% to 65%, which utilizes adiketopiperazine having the formula3,6-bis(N—X-4-aminobutyl)-2,5-diketopiperazine disodium salt ormagnesium salt, wherein X is selected from the group consisting offumaryl, succinyl, maleyl, and glutaryl. In an exemplary embodiment, thediketopiperazine has the formula(bis-3,6-(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine or3,6-bis(N-fumaryl-4-amino-butyl)-2,5-diketopiperazine.

Another embodiment disclosed herein includes a method of delivering adrug, for example, a peptide such as insulin to a patient in needthereof comprising administering a dry powder to the deep lung byinhalation of the dry powder by the patient; wherein the dry powdercomprises diketopiperazine microparticles comprising insulin, a zincsalt and citrate; wherein the microparticles are formed of adiketopiperazine and have a SSA ranging from about 35 to about 67 m²/gor about 40 to about 67 m²/g and/or in microparticles having a transisomer content raging from about 45% to about 65%. In aspects of thisembodiment, particular features of an inhaler system are specified.Further embodiments involve methods of treating an insulin-relateddisorder comprising administering a dry powder described above to aperson in need thereof. In various embodiments an insulin-relateddisorder can specifically include or exclude any or all of pre-diabetes,type 1 diabetes mellitus (honeymoon phase, post-honeymoon phase, orboth), type 2 diabetes mellitus, gestational diabetes, hypoglycemia,hyperglycemia, insulin resistance, secretory dysfunction, impairedearly-phase release of insulin, loss of pancreatic β-cell function, lossof pancreatic β-cells, and metabolic disorder.

One embodiment comprises a method of treating an endocrine-relateddisease or disorder comprising administering to a person in need thereofa dry powder formulation comprising a peptide hormone, including,oxytocin, GLP-1 and the like, citrate and a divalent cationic salt. Inone embodiment, the dry powder formulation can comprise disodium FDKPmicroparticles or FDKP microparticles having a specific surface area ofless than about 67 m²/g and a drug suitable to treat said disease ordisorder. Further embodiments include a method of treating aninsulin-related disorder comprising administering a dry powdercomprising microparticles of disodium FDKP or FDKP, a citrate, acetateor tartrate buffer and a divalent cation, including zinc, magnesium andcalcium or a monovalent cation, including, sodium, potassium and lithiumdescribed above to a person in need thereof. The method can compriseadministering to a subject a dry powder formulation. In variousembodiments, a hormone-related disorder such as post-partum hemorrhage,or any other oxytocin-related disease can be treated with theformulation comprising oxytocin. In embodiments wherein aninsulin-related disorder is to be treated, the formulation comprisinginsulin the subject to be treated can specifically include or excludeany or all of pre-diabetes, type 1 diabetes mellitus (honeymoon phase,post-honeymoon phase, or both), type 2 diabetes mellitus, gestationaldiabetes, hypoglycemia, hyperglycemia, insulin resistance, secretorydysfunction, impaired early-phase release of insulin, loss of pancreaticβ-cell function, loss of pancreatic β-cells, and metabolic disorder. Inone embodiment, the dry powder comprises insulin. In other embodiments,the dry powder comprises glucagon, an exendin, or GLP-1, PTH, PTHrP,combinations thereof, and the like.

In embodiments herewith, specific RF/fill values can depend on theinhaler used to deliver the powder. Powders generally tend toagglomerate and crystalline DKP microparticles form particularlycohesive powders. One of the functions of a dry powder inhaler is todeagglomerate the powder so that the resultant particles comprise arespirable fraction suitable for delivering a dose by inhalation.However, deagglomeration of cohesive powders is typically incomplete sothat the particle size distribution seen when measuring the respirablefraction as delivered by an inhaler will not match the size distributionof the primary particles, that is, the profile will be shifted towardlarger particles. Inhaler designs vary in their efficiency ofdeagglomeration and thus the absolute value of RF/fill observed usingdifferent designs will also vary. However, optimal RF/fill as a functionof specific surface area will be similar from inhaler to inhaler.

One class of drug delivery agents that has been used to overcomeproblems in the pharmaceutical arts such as drug instability and/or poorabsorption are the 2,5-diketopiperazines. 2,5-Diketopiperazines can beformed into microparticles that incorporate a drug or microparticlesonto which a drug can be adsorbed. The combination of a drug and adiketopiperazine can impart improved drug stability and/or absorptioncharacteristics. These microparticles can be administered by variousroutes of administration. As dry powders these microparticles can bedelivered by inhalation to specific areas of the respiratory system,including the lungs.

Such microparticles are typically obtained by pH-based precipitation ofthe free acid (or base) resulting in self-assembled microparticlescomprised of aggregated crystalline plates. The stability of theparticle can be enhanced by small amounts of a surfactant, such aspolysorbate-80, in the DKP solution from which the particles areprecipitated (see for example US Patent Publication No. 2007/0059373entitled “Method of drug formulation based on increasing the affinity ofcrystalline microparticle surfaces for active agents” which isincorporated herein by reference in its entirety for all that it teachesregarding the formation and loading of DKP microparticles and drypowders thereof). Ultimately solvent can be removed to obtain a drypowder. Appropriate methods of solvent removal include lyophilizationand spray drying (see for example US Patent Publication No. 2007/0196503entitled “A method for improving the pharmaceutic properties ofmicroparticles comprising diketopiperazine and an active agent” and U.S.Pat. No. 6,444,226 entitled “Purification and stabilization of peptideand protein pharmaceutical agents” each of which is incorporated hereinby reference in its entirety for all that it teaches regarding theformation and loading of DKP microparticles and dry powders thereof).The microparticles disclosed herein can be composed of DKP free acid orbases or composed of DKP salts. Such particles are typically formed (asopposed to dried) by spray drying, resulting in spheres and/or collapsedspheres of an amorphous salt (as opposed to a free acid or base) so thatthey are chemically, physically, and morphologically distinct entities.In embodiments the present disclosure refers to FDKP as the free acid orthe dissolved anion. In embodiments herewith, an exemplary embodimentincludes the disodium salt of FDKP or FDKP disodium salt as disclosedand contemplated in U.S. Pat. Nos. 7,820,676 and 8,278,308, which areincorporated herein by reference in its entirety.

Methods for synthesizing diketopiperazines are described in, forexample, Katchalski, et al., J. Amer. Chem. Soc. 68, 879-880 (1946) andKopple, et al., J. Org. Chem. 33(2), 862-864 (1968), the teachings ofwhich are incorporated herein by reference for all they discloserelating to diketopiperazine synthesis.2,5-Diketo-3,6-di(aminobutyl)piperazine (Katchalski et al. refer to thisas lysine anhydride) can also be prepared via cyclodimerization ofN-ε-P-L-lysine in molten phenol, similar to the Kopple method, followedby removal of the blocking (P)-groups with an appropriate reagent andconditions. For example, CBz-protecting groups can be removed using 4.3M HBr in acetic acid. This route can be preferred because it uses acommercially available starting material, it involves reactionconditions that are reported to preserve stereochemistry of the startingmaterials in the product and all steps can be easily scaled up formanufacture. Methods for synthesizing diketopiperazines are alsodescribed in U.S. Pat. No. 7,709,639, entitled, “Catalysis ofDiketopiperazine Synthesis,” which is also incorporated by referenceherein for its teachings regarding the same.

Fumaryl diketopiperazine(3,6-bis(N-fumaryl-4-aminobutyl)-2,5-diketopiperazine; FDKP) is onepreferred diketopiperazine for pulmonary applications:

FDKP provides a beneficial microparticle matrix because it has lowsolubility in acid but is readily soluble at neutral or basic pH. Theseproperties allow FDKP to crystallize and the crystals to self-assembleinto form microparticles under acidic conditions. The particles dissolvereadily under physiological conditions where the pH is neutral. Asnoted, microparticles having a diameter of between about 0.5 and about10 microns can reach the lungs, successfully passing most of the naturalbarriers. Particles in this size range can be readily prepared fromFDKP.

As noted, microparticles having a diameter of between about 0.5 andabout 10 microns can reach the lungs, successfully passing most of thenatural barriers. Particles in this size range can be readily preparedfrom diketopiperazines with acidic groups, such as the carboxylategroups in FDKP (as well as in related molecules such as2,5-diketo-3,6-di(4-X-aminobutyl)piperazine wherein X is succinyl,glutaryl, or maleyl). Upon acid precipitation self-assembled particlescomposed of aggregates of crystalline plates are obtained. The size ofthese plates relates to the specific surface area of the particles whichin turn is implicated in effects on the structure, loading capacity, andaerodynamic performance of the particles.

The SSA of DKP microparticles is a measure of average crystal size andcan be used to gauge the relative contributions of crystal nucleationand growth to microparticle characteristics. SSA depends on the size ofmicroparticle crystals and the density (p) of the microparticle matrixand is inversely proportional to the characteristic size, L, of thecrystals. Embodiments disclosed herein show that microparticles with aspecific surface area less than about 67 m²/g exhibit characteristicsbeneficial to delivery of drugs to the lungs such as improvedaerodynamic performance with moderately efficient inhalers such as theMEDTONE® inhaler disclosed in U.S. Pat. No. 7,464,706 entitled, “UnitDose Cartridge and Dry Powder Inhaler,” which is incorporated byreference herein for its teachings regarding the same. An alternateembodiment with a specific surface area less than about 62 m²/g providesa greater level of assurance that a batch of particles will meet aminimum aerodynamic performance standard. As SSA also affects drugloading/content capacity, various embodiments require SSA 40, or 45 m²/gfor improved drug adsorption capacity. Additionally, as SSA falls belowabout 35 m²/g inconsistent cartridge emptying is observed even with highefficiency inhalers such as those disclosed in U.S. patent applicationSer. No. 12/484,125 (now U.S. Pat. No. 8,499,757, entitled, “A DryPowder Inhaler and System for Drug Delivery,” filed on Jun. 12, 2009,and U.S. patent application Ser. No. 12/717,884, now U.S. Pat. No.8,485,180, entitled, “Improved Dry Powder Drug Delivery System,” filedon Mar. 4, 2010, which disclosures are herein incorporated by referencefor its teachings regarding the same.

FDKP Microparticle Formation.

The first step in the manufacture of FDKP microparticles is theformation of the microparticles by pH-induced crystallization of FDKPand the self-assembly of the FDKP crystals into microparticles having anoverall spherical morphology (FIG. 2). Accordingly, the manufacture ofmicroparticles is essentially a crystallization process. Excess solventcan be removed by washing the suspension by repeated centrifugation,decantation and re-suspension, or by diafiltration.

In one embodiment, to form peptide-loaded FDKP microparticles, forexample, insulin can be adsorbed directly onto the microparticles whilein suspension (i.e. prior to freeze drying) by adding an insulin stocksolution to the FDKP microparticle suspension comprising a citratebuffer. In embodiments, a pH control step can also be performed afterthe addition of the insulin stock solution. This step can promoteinsulin adsorption onto the microparticles in suspension prior tofurther processing. Increasing the pH of the suspension to about 4.5promotes complete insulin adsorption onto the microparticles insuspension without excessive dissolution of the FDKP from the particlematrix and also improves the stability of insulin in the bulk drugproduct. The suspension can be flash-frozen drop-wise (i.e.cryo-pelletized) in liquid nitrogen and lyophilized to remove thesolvent and obtain a dry powder. In alternative embodiments thesuspension can be spray-dried to obtain the dry powder.

In one embodiment, a manufacturing process for making the present FDKPmicroparticles containing insulin is provided. In summary, using a highshear mixer such as a Dual-feed SONOLATOR™ at 2000 psi through a0.001-in² orifice, or for example, the high shear mixer as disclosed inU.S. Provisional Patent Application Ser. No. 61/257,311, filed on Nov.2, 2009, which disclosure is incorporated herein by reference in itsentirety for all that it teaches regarding the production of DKPmicroparticlesparticles, equal masses of about 10.5 wt % acetic acid andabout 2.5 wt % FDKP solutions at about 16° C.±about 2° C. can be fed at2000 psi through a 0.001-in² orifice. The precipitate can be collectedin a deionized (DI) water reservoir of about equal mass and temperature.The resultant suspension comprises about 0.8% solids. The precipitatecan be concentrated and washed by tangential flow filtration. Theprecipitate can be first concentrated to about 4% solids then washedwith deionized water. The suspension can be finally concentrated toabout 10% solids based on the initial mass of FDKP. The concentratedsuspension can be assayed for solids content by an oven drying method.In this embodiment, the FDKP microparticles in suspension arehomogenized with zinc and citrate solution containing the insulin toform the powder particles then sprayed dried or lyophilized.

The microparticles described herein can comprise one or more activeagents. As used herein “active agent”, used interchangeably with “drug”,refers to pharmaceutical substances, including small moleculepharmaceuticals, biologicals and bioactive agents. Active agents can benaturally occurring, recombinant or of synthetic origin, includingproteins, polypeptides, peptides, nucleic acids, organic macromolecules,synthetic organic compounds, polysaccharides and other sugars, fattyacids, and lipids, and antibodies and fragments thereof, including, butnot limited to, humanized or chimeric antibodies, F(ab), F(ab)₂, asingle-chain antibody alone or fused to other polypeptides ortherapeutic or diagnostic monoclonal antibodies to cancer antigens. Theactive agents can fall under a variety of biological activity andclasses, such as vasoactive agents, neuroactive agents, hormones,anticoagulants, immunomodulating agents, cytotoxic agents, antibiotics,antiviral agents, antigens, infectious agents, inflammatory mediators,hormones, and cell surface antigens. More particularly, active agentscan include, in a non-limiting manner, cytokines, lipokines,enkephalins, alkynes, cyclosporins, anti-IL-8 antibodies, IL-8antagonists including ABX-IL-8; prostaglandins including PG-12, LTBreceptor blockers including LY29311, BIIL 284 and CP105696, triptanssuch as sumatriptan and palmitoleate, insulin and analogs thereof,growth hormone and analogs thereof, parathyroid hormone (PTH) andanalogs thereof, parathyroid hormone related peptide (PTHrP), ghrelin,obestatin, enterostatin, granulocyte macrophage colony stimulatingfactor (GM-CSF), amylin, amylin analogs, glucagon-like peptide 1(GLP-1), Texas Red, clopidogrel, PPACK(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), oxyntomodulin(OXM), peptide YY(3-36) (PYY), adiponectin, cholecystokinin (CCK),secretin, gastrin, glucagon, motilin, somatostatin, brain natriureticpeptide (BNP), atrial natriuretic peptide (ANP), IGF-1, growth hormonereleasing factor (GHRF), integrin beta-4 precursor (ITB4) receptorantagonist, nociceptin, nocistatin, orphanin FQ2, calcitonin, CGRP,angiotensin, substance P, neurokinin A, pancreatic polypeptide,neuropeptide Y, delta-sleep-inducing peptide and vasoactive intestinalpeptide.

The drug content to be delivered depends on the need of the subject andthe potency of the drug. In certain embodiments, microparticles formedfrom FDKP having a trans isomer content between 45% and 65% is typicallygreater than 0.01% are used. In one embodiment, the drug content to bedelivered with the microparticles having the aforementioned trans isomercontent, can range from about 0.01% to about 20%, which is typical forpeptides such as insulin. For example, if the drug is insulin, thepresent microparticles typically comprise 3-6 U/mg (approximately 10 to15%) insulin. In certain embodiments, the drug content of the particlescan vary depending on the form and size of the drug to be delivered.

The range of loading of the drug to be delivered is typically betweenabout 0.01% and about 90%, depending on the form and size of the drug tobe delivered and the potency of the dose required. For oxytocin,preferred loads are about 0.5% to about 50% (w/w); or from about 0.5%(w/w) to about 20% (w/w).

As long as the DKP microparticles described herein retain the requiredisomer content, they can adopt other additional characteristicsbeneficial for delivery to the lung and/or drug adsorption. U.S. Pat.No. 6,428,771 entitled “Method for Drug Delivery to the PulmonarySystem” describes DKP particle delivery to the lung and is incorporatedby reference herein for its teachings regarding the same. U.S. Pat. No.6,444,226, entitled, “Purification and Stabilization of Peptide andProtein Pharmaceutical Agents” describes beneficial methods foradsorbing drugs onto microparticle surfaces and is also incorporated byreference herein for its teachings regarding the same. Microparticlesurface properties can be manipulated to achieve desired characteristicsas described in U.S. patent application Ser. No. 11/532,063, now U.S.Pat. No. 7,799,344, entitled “Method of Drug Formulation based onIncreasing the Affinity of Crystalline Microparticle Surfaces for ActiveAgents” which is incorporated by reference herein for its teachingsregarding the same. U.S. patent application Ser. No. 11/532,065,entitled “Method of Drug Formation based on Increasing the Affinity ofActive Agents for Crystalline Microparticle Surfaces” describes methodsfor promoting adsorption of active agents onto microparticles. U.S.patent application Ser. No. 11/532,065, now U.S. Pat. No. 7,803,404 isalso incorporated by reference herein for its teachings regarding thesame.

EXAMPLES

The following examples are included to demonstrate embodiments of thedisclosed microparticles. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the present disclosure, and thus can be considered toconstitute preferred modes for its practice. However, those of ordinaryskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments which aredisclosed and still obtain a like or similar result.

Example 1

Preparation, Characterization and Stability of Oxytocin Spray-DriedPowders

Fourteen powders containing 1% (w/w) oxytocin and varying amounts ofbuffers, salts, carriers, excipients, including, trehalose, PVP,isoleucine, cystine, trileucine, FDKP, sodium citrate and zinc salt,obtained from various vendors as described in Table 1 below, wereprepared at the 7 g scale as shown on Table 2 below. The samples wereprepared by weighing the amounts required as stated in Table 2 anddissolved in deionized water to form a solution or suspension, theoxytocin was added and mixed prior to spray drying. In the samples usingthe citrate buffer and the divalent cation, oxytocin was dissolved inthe citrate buffer prior to adding the rest of the ingredients in themixture. The solution or suspension was then spray-dried using theparameters as described in Table 3 below. Suspensions were homogenizedin a high shear mixer prior to spray drying. The solutions were filteredthrough a 0.2 μm membrane prior to spray-drying.

The dry powders were collected and used in the experiments describedbelow. Experiments were conducted to characterize the powders obtainedusing various techniques, including to measure the oxytocin content ofvarious samples before and after incubation to determine yields, loss ondrying (LOD), aerodynamic performance, particle size and particlemorphology were evaluated. Stability studies were carried out usingaliquots from each of the dry powder samples prepared, which had beenincubated at 40° C. in a relative humidity of 75% (40° C./75% RH) inscintillation vials sealed with a fluoropolymer resin lined screw capwhich had been placed in heat-sealed aluminum pouches for the timeperiods studied. Samples of the incubated material were taken at varioustimes after onset of the experiments and up to approximately 7 months.The samples were evaluated by high performance liquid chromatography(HPLC) assay (see preparation described below) to determine the presenceof the oxytocin in the samples and the degradation products. Oxytocinstability studies were performed up to 11 months for certain powders,including, Sample ID Nos. 4, 6 and 13 in Table 2.

TABLE 1 Formulation Components Chemicals Supplier FDKP Na MannKind PVPK30 Spectrum Trehalose Alfa Aesar L-isoleucine Alfa Aesar TrileucineBachem Cystine Alfa Aesar Citric acid anhydrous EMD Trisodium citratedihydrate Alfa Aesar Zinc chloride Sigma Aldrich Zinc citrate SigmaAldrich Oxytocin American peptide Büchi mini spray dryer B 290Filtration unit fast PES membrane (0.2 μm) 150 mL system (Nalgene)Homogenizer (Tekmar Tissumizer)

TABLE 2 Sample Formulations Contents Sample Wt. % Target (dry basis) IDNo. Citrate ZnCl₂ Trehalose Na₂FDKP ILE* PVP 1 — — 87.00 — 10.00 2.00 224.03 6.76 59.94 — 6.89 1.38 3 12.01 3.38 73.47 — 8.45 1.69 4  4.81 1.3581.59 — 9.38 1.88 5 24.03 — 65.89 — 7.57 1.51 6 27.73 6.76 56.69 — 6.521.30 7 27.73 6.76 38.44 19.55 6.52 — 8 — — 59.00 30.00 10.00 — 9 — —44.00 44.00 10.00 1.00 10 — — 89.00 — 10.00 — 11 — — 87.00 — TLE* 2.0010.00 12 — — 87.00 — CYS* 2.00 10.00 13 24.03 Zn citrate 39.26 — ILE0.90 30.30 4.51 *ILE: isoleucine, TLE: trileucine, CYS: cystine.

Spray-dried powders were prepared with a target oxytocin content of 1%.Formulation contents are detailed in Table 2. A mixture having a ratioof 87:10:2 by weight of trehalose, isoleucine and PVP served as a matrixfor control formulations of Samples ID Nos. 1 to 6. To this mixture wereadded sodium citrate and zinc. The quantities of citrate salts werevaried from 100 to 20 equivalents per mole of oxytocin (24 to 4.8% oftotal weight). The quantities of the zinc salts were varied from 50 to10 equivalents per mole of oxytocin (6.7 to 1.3% of total weight).Concentrated sodium citrate buffers (75 mM pH 4.5 and 6.5) were used asthe source of citrate.

The solids content of the feed solutions was kept constant at 5%. Feedsolutions were filtered prior spray-drying. One formulation containingFDKP appeared cloudy and was not filtered (Sample ID No. 7); mixturescontaining trileucine, cystine and zinc citrate (Samples ID No. 11, 12and 13) required homogenization and the resulting suspensions were keptunder constant stirring during the spray-drying process. A suspensioncontaining zinc citrate and citrate buffer was prepared as feed solutionand homogenized in a high sheer mixing (Tissumizer homogenizer). Asecond solution containing oxytocin and the remaining excipients inwater were added to the suspension and the final weight was adjusted to140 g with deionized water.

TABLE 3 Spray-drying conditions Inlet: 130° C. Outlet: 63° C. Drying gasflow: 60 mbar (nitrogen) Atomization flow: ~59.9 g/h Aspirator 80% Pump 5%

The oxytocin stability study results from are shown in Table 4 below.The data is shown as the percent (%) of oxytocin remaining in the samplecompared to the starting amount of material used. As seen in Table 4 andFIG. 3, three of the powder formulations (Sample ID Nos. 4, 6, and 13)tested maintained more than about 90% of the oxytocin as assayed after32 weeks of incubation. The data also show that the combination ofsodium citrate and zinc salt led to the highest stability (lessdegradation of oxytocin) in solid or dry powder form (about 100%, SampleID No. 6). Moreover, the addition of sodium citrate and zinc also led tohigher respirable fraction per fill content of powder (RF/fill) used,with a maximum RF/fill of 60.2% for a powder containing 12% (w/w) sodiumcitrate, 3.4% (w/w) zinc chloride, 73.5% (w/w) trehalose, 8.4% (w/w)isoleucine and 1.69% (w/w) PVP. The control powder formulated withoutzinc and citrate, had a RF/fill of 40.9% (Sample ID No. 1), but itsoxytocin degradation rate was more rapid as there was only 51.6%oxytocin remaining in the sample after 32 weeks of incubation.

Scanning electron micrographs (SEM) of sample control powders werestudied and shown in FIG. 1A (low magnification) and FIG. 1B. (highmagnification The SEMs show regularly-shaped, substantially sphericalparticles, which appear substantially homogeneous in size with smallsurface indentations and typical of amorphous powders.

TABLE 4 Stability results for oxytocin powders at 40° C./75% RH ID No. t= 0 t = 2 W t = 4 W t = 8 W t = 12 W t = 16 W t = 20 W t = 24 W t = 28 Wt = 32 W t = 36 W t = 40 W t = 44 W 1 100 87.8 68.3 67.6 69.1 69.5 59.861.3 57.0 51.6 2 100 90.3 87.1 86.4 83.2 81.9 79.9 77.8 12.7 75.7 3 10091.9 89.8 88.2 86.8 84.6 79.3 81.1 78.1 79.1 4 100 94.1 95.8 94.0 94.189.5 90.0 87.3 90.2 89.2 82.8 82.3 81.6 5 100 98.1 94.0 93.5 88.8 87.084.9 80.5 82.5 81.6 6 100 99.1 97.8 97.8 93.2 95.6 94.9 91.5 92.9 101.4101.4 89.4 90.0 7 100 91.4 90.1 88.9 86.5 78.2 73.2 70.1 73.0 67.6 8 10095.9 91.1 86.4 55.3 71.7 60.7 71.7 63.4 60.3 9 100 94.9 94.1 92.5 88.082.2 81.3 78.3 74.2 66.1 10 100 89.6 85.6 67.3 59.8 51.1 50.0 49.1 46.446.6 11 100 99.3 89.5 88.2 83.5 77.5 73.8 76.2 73.6 72.4 12 100 94.493.1 87.0 84.5 78.9 83.7 82.0 79.1 76.4 13 100 98.8 94.2 93.3 91.0 91.789.5 90.3 92.5 92.0 93.6 87.6 83.2Dry Powder Characterizations: LOD), RF/Fill, SEM, and Oxytocin Assay

Loss on drying (LOD) was measured by thermogravimetric analysis (TGA)with a heat and hold method (20° C./min, 110° C. isotherm for 30minutes). The powders were obtained with an average yield of 73.8% and aminimum LOD of 4.63%.

Aerodynamic performance of the spray-dried powders was measured byAndersen Cascade Impaction with the Gen2C inhaler (30 Lpm, 8s, MannKindCorp.) and the results are shown in Table 5. Geometric particle size wasdetermined by laser diffraction using a Sympatec RODOS M powderdisperser set at 0.5 bar and 3 bar dispersing pressures. Particlemorphology was assessed by field emission scanning electron microscopy.Table 5 shows that particles range in size from about 3.8 to 5.6 μm at0.5 bar and 3 bar atmospheric pressures tested and had a % RF/fill ofabout 40 to about 60%. As see in Table 5, the samples containing citrateand zinc (Sample ID No. 2, 3 and 4) performed best as shown by thecartridge emptying data (>70%) and emitted dose of 56% to 60%. Densityof the powders was evaluated with a tapped density analyzer (Autotap)after 3000 taps. Density of the bulk powder didn't exceed 0.5 g/mlregardless of salt contents.

Oxytocin content was evaluated using an HPLC method. Oxytocin standardsolutions were prepared at approximately 250 μg/mL in 0.1M sodiumbicarbonate pH 9.5 (6.25 mg of oxytocin raw material in 25.0 mL).Powders were prepared by dissolving 10±1.0 mg in 0.1M sodium bicarbonatepH 9.5 to give a final oxytocin concentration of 0.250 mg/mL. Initialdrug content was assayed to ascertain the starting material. Powderswere prepared with a target drug content of 1% and assays confirmed theoxytocin content between 0.92 and 1.13%.

Oxytocin Stability in Powder Form

The powders were weighed into 20 mL glass vials that were then closed,wrapped in foil, and heat sealed. The foil pouches were placed on astability chamber at 40° C./75% RH. Samples were pulled at 2 and 4weeks; then pulled every 4 weeks and up to 32 weeks after incubation.Samples were stored frozen (−20° C.) until assayed by HPLC as discussedabove.

TABLE 5 Aerodynamic performance, particle size and densitycharacterizations Aerodynamic performance Particle size Density (30 LPM,8 s) (μm) (g/l) ID No. % RF % RF/fill % CE 0.5 bar 3 bar D_(Bulk)D_(Tap) 1 58.4 40.9 70.0 4.06 3.85 0.411 0.553 2 76.5 59.1 77.2 4.093.86 0.472 0.594 3 77.5 60.3 77.8 4.13 3.98 0.408 0.582 4 71.4 55.4 77.34.12 4.07 0.456 0.623 5 74.3 55.9 75.3 4.18 3.96 0.454 0.649 6 69.4 59.886.2 4.22 4.16 0.389 0.620 7 72.8 42.5 58.3 5.62 5.61 — — 8 — — — 4.104.02 — — 9 — — — 4.38 4.29 — — 10 68.8 59.5 86.4 4.05 3.86 — — 11 56.450.6 89.6 5.25 4.90 — — 12 — — — 4.07 3.83 — — 13 70.8 53.2 75.1 4.174.00 0.397 0.580

Aerodynamic Performance, Particle Size and Morphology

Aerodynamic testing on selected powders highlighted the beneficialeffect of combining sodium citrate and zinc with trehalose, isoleucineand PVP. The effect was observed with citrate and zinc contents as lowas 4.8% and 1.3% respectively (Table 5, Sample ID No. 4). The maximumeffect (60.3% RF/fill) was obtained with 12% citrate content and 3.4%zinc. Powder Sample ID No. 2, prepared with twice the amount of citrateand zinc, had an RF/fill of 59.1%. Both Sample powders ID Nos. 2 and 4when tested in the inhaler were delivered out of the inhaler at about77% of the original content. The RF/fill of the control powder (SampleID No. 1) formulated with trehalose, isoleucine and PVP (87/10/2) was40.9% and at a rate less than the samples containing zinc and citrate.

Particle morphology studied by scanning electron microscopy shows thatspray-drying of the control formulation (Sample ID No. 1) containingtrehalose, PVP and isoleucine produced slightly corrugated, sphericalparticles typical of leucine-containing powders (FIG. 2A). Thecorrugated substantially spherical morphology was maintained with theaddition of salts (zinc and citrate salts) to the mixture containingtrehalose, PVP and isoleucine (FIGS. 2B; 2C and 2D). However, theparticles containing zinc and citrate differ from the controls as theyappear slightly more corrugated and less spherical. As shown in theSEMs, the particles formed with zinc and/or citrate appear substantiallyspherical and have a slightly more indentations, corrugated surface orwrinkle appearance, and less regular pattern. It was observed that theparticles containing oxytocin, zinc and citrate appeared to be morefragile or more collapsible than the controls during preparation inparticular during vacuum drying step.

FIG. 4 provides a graphic representation of data obtained from X-raydiffraction studies of dry powders showing amorphous content of thepowders by their characteristic scan patterns. The data demonstratesthat the X-ray diffraction analysis confirmed that the spray-driedpowders all appear as uniform amorphous in content as demonstrated bydata scans depicted in FIG. 4.

The data also demonstrates that the addition of citrate/zinc to a powdercontaining FDKP (Sample ID No. 7) (19.5% w/w), trehalose (38.4% w/w) andisoleucine (6.5% w/w) produced a powder with improved properties (42%RF/fill) over the powder without citrate and zinc. The powder containingcitrate yielded a 17% improvement in RF/fill over a powder formulatedwithout citrate/zinc (25.6% RF/fill, 78.4% CE).

The present powders were not excessively cohesive because their mediangeometric particle sizes were similar at 0.5 bar and 3 bar RODOSdispersing pressures. The average values were 4.34 and 4.18 μm at 0.5and 3 bars.

The data in Table 5 show the aerodynamic performance of the powders.Table 5 shows that the powders containing citrate and zinc yielded highrespirable fractions (>70%) and cartridge emptying data in someinstances were greater than >90% (data not shown). Sample testing in ananatomically correct airway model showed that about 73% of the dose inan inhaler containing the powders is delivered to the lungs.

Oxytocin Stability Studies

The data indicate that out of the fourteen powders prepared, threemaintained more than 89% of the original total oxytocin content obtainedafter 32 weeks of incubation at 40° C./75% RH (Table 4). The degradationrate appears to be the highest before 4 weeks for the most stablepowders (FIG. 3) then plateaus; this early onset of degradation isprobably due to moderate to high residual water content in the powders.The most stable powders was prepared from a pH 6.5 citrate buffer andzinc chloride as a source of zinc divalent cations. Overall, powdersprepared in presence of both citrate and zinc salts exhibited thehighest stability. The stabilizing effect of this combination was evenobserved at low salts contents (Sample ID No. 1 vs. Sample ID Nos. 2, 3,4). Among the “non-buffered” formulations, the addition of disodium FDKPor replacing isoleucine by trileucine or cystine enhanced the stabilityof the powders.

Example 2

Preparation, Characterization and Stability of Alternative OxytocinSpray-Dried Powder Embodiments

Preparation of oxytocin spray-dried powders was performed as in Example1 above. In these experiments, fourteen powders containing 1% (w/w)oxytocin and varying amounts of buffers, salts, carries excipients,including, trehalose, PVP, isoleucine, sodium citrate, citric acid,sodium tartrate, tartaric acid and zinc salt, obtained from variousvendors as described in Table 6 below, were prepared at the 2.5 g scaleas shown on Table 1 below. In these experiments, L-(+)-tartaric acid andsodium L-(+)-tartrate dihydrate were used and obtained from Alfa Aesar.Unlike in Example 1, bulk solid sodium citrate salt and citric acid wereused as source of sodium citrate. Samples containing 1% (w/w) oxytocinwere made as described in Example 1 and the solutions or suspensionswere then spray-dried using the parameters as described in Table 6below.

The dry powders were collected and used in the experiments describedbelow. Powders identified with sample numbers ID Nos. 14 to 28 wereobtained with an average yield of 76.7% and a minimum loss on drying(LOD) of 5.73% (measured by Karl Fisher titration). Powders ID Nos. 14to 28 were submitted to an additional drying step under vacuum pump thatled to a minimum LOD of 2.90%.

Spray-dried powders containing a target oxytocin content of 1% wereassayed and the data confirmed the oxytocin values ranged between 0.87to 1.01%. The components of the prepared formulations are detailed inTable 7 showing the contents of each sample made and tested. In certainembodiments, a mixture having a ratio of 87:10:2 by weight of trehalose,isoleucine and PVP served as a matrix for all the formulations exceptsamples ID No. 20 and 21. To this mixture sodium citrate, citric acidand zinc were added. The quantities of citrate salts were varied from100 to 50 equivalents per mole of oxytocin (29.2 to 14.6% of totalweight). The quantities of the zinc salts were varied from 50 to 5equivalents per mole of oxytocin (30.3 to 0.7% of total weight). In someembodiments, the zinc cation appeared to be essential to the compositioncharacteristics as exemplified by zinc chloride use alone (Sample ID No.22) also provided improved stability of the powders.

TABLE 6 Drying Conditions Inlet: 150° C. Outlet: 70° C. Drying gas flow:60 mbar (nitrogen) Atomization flow: ~59.9 g/h Aspirator 80% Pump  5%

TABLE 7 Oxytocin Sample compositions Wt. % Target (dry basis) SampleSodium Citric ID No. citrate acid ZnCl₂ Trehalose ILE PVP 14 29.2 — 6.856.3 6.5 1.3 15 29.2 — 2.7 59.8 6.9 1.4 16 29.2 — 1.4 61.0 7.0 1.4 1729.2 — 0.7 61.6 7.1 1.4 18 14.6 — 6.8 69.1 7.9 1.6 19 24.3 3.16 6.8 57.86.6 1.3 20 29.2 — 6.8 64.0 — — 21 — — 6.8 57.6 6.4 — 22 — — 6.8 81.94 9.42  1.88 Zinc citrate 23 — — 30.3 61.2 7.0 1.4 24 — — 9.1 79.9 9.21.8 25 24.3 3.16 30.30 37.07 4.26 0.85 26 14.6 — 30.30 48.42 5.57 1.11Sodium Tartaric tartrate acid 27 22.8 — 6.8 61.9 7.1 1.4 28 19.0 3.0 6.862.6 7.2 1.4

Aerodynamic Performance of Dry Powders

Aerodynamic performance of the spray-dried powders was measured byAndersen Cascade Impaction with the Gen2C inhaler (21.6 Lpm, 4s,MannKind Corp.) and the results are shown in Table 8. High % Rf/fill(>50%) were obtained even under a low peak inspiratory pressure. Thedata in Table 8 Shows the % RF/fill ranging from about 20 to about 60%and cartridge emptying of total contents was up to 77% (Sample ID No.22). The highest % Rf/Fill were obtained for powders containing zinccitrate, zinc chloride with or without PVP. As seen in Table 8, %Rf/fill was improved by the addition of isoleucine (Sample ID Nos. 14and 20). The samples containing zinc citrate or zinc chloride alone(Sample ID Nos. 22, 23 and 24) had high % Rf/fill of about 50 to 60% andcartridge emptying greater than 70%.

Aerodynamic testing on selected powders highlighted the beneficialeffect of combining sodium citrate and zinc chloride with trehalose,isoleucine with or without PVP as exemplified by Sample ID No. 18, 19,20 and 21. The improved performance of the powders was observed withcitrate and zinc contents as low as 14.6% and 1.4% respectively (SampleID Nos. 16 and 18). The maximum effect (53.0% RF/fill) was obtained with14.6% content in sodium citrate and 6.8% in zinc (Sample ID No. 18). Thebeneficial effect of combining zinc citrate with trehalose, isoleucineand PVP is exemplified by the performance of powders Sample ID Nos. 23and 24, which yielded % Rf/fill greater than 50% and cartridge emptyingof about 73%.

TABLE 8 Aerodynamic performance by Andersen cascade impactor. Sample IDNo. % RF/fill % CE 14 43.3 57.2 15 47.3 62.1 16 49.8 61.2 17 22.4 35.318 53.0 71.9 19 44.8 61.6 20 36.0 52.4 21 50.3 66.9 22 51.2 77.0 23 51.573.0 24 60.3 72.7 25 37.5 55.0 26 37.7 59.3 27 57.3 74.7 28 45.1 59.6

Oxytocin Formulation Stability Studies

Stability of oxytocin spray-dried powders was performed as in Example 1above. Stability testing was performed up to 40 weeks. The oxytocinstability study results from the assays are shown in Table 9 below andFIG. 5. The data is shown as the percent (%) remaining of samplecompared to the starting material used. As seen in Table 9, only 3 ofthe powder formulations (Sample ID Nos. 16, 27, and 28) testedmaintained less than about 90% of the oxytocin when aliquots of thesample were assayed after 40 weeks incubation. The combination ofcitrate and zinc salts led to the highest stability in solid state(greater than about 90%). The highest stability was achieved with thecombination containing 14.6% sodium citrate and 6.8% zinc chloride(Sample ID No. 18). Powders containing a minimum 9.1% content in zinccitrate with or without sodium citrate maintained more than 97% of theoxytocin after 40 weeks incubation. FIG. 5 is a graphic representationof dry powder samples from the stability studies wherein the samplescontaining divalent zinc salt and citrate salts at variousconcentrations showed a slow degradation of the oxytocin over a periodof 40 weeks; wherein the samples tested retained greater than 90% of theoxytocin content.

In the alternative embodiment using tartrate, powders containing zincand tartrate salts maintained also about 90% of the oxytocin contentafter 24 weeks of incubation and greater than 85% of the oxytocincontent after 32 weeks of sample incubation.

TABLE 8 Stability results for oxytocin powders at 40° C./75% RH ID No. t= 0 t = 2 W t = 4 W t = 8 W t = 14 W t = 16 W t = 20 W t = 24 W t = 32 Wt = 40 W 14 100 99.9 101.9 100.0 99.1 94.6 100.9 83.5 85.8 91.4 15 10098.5 104.2 97.8 97.5 96.0 93.5 78.0 74.3 95.7 16 100 100.2 102.2 94.996.0 100.8 55.1 79.7 41.7 33.9 17 100 97.0 99.7 96.7 96.5 93.7 94.3 91.185.3 90.1 18 100 105.2 109.5 103.3 107.0 104.2 103.2 101.4 103.1 103.819 100 103.5 102.2 102.4 102.1 101.5 101.6 92.8 94.6 96.9 20 100 103.5104.5 103.1 103.2 103.3 105.1 93.3 101.4 96.0 21 100 100.2 100.9 101.2101.2 101.2 99.6 99.3 93.3 92.9 22 100 101.4 100.5 98.0 96.3 100.1 95.795.6 92.4 94.2 23 100 99.6 97.6 100.1 99.0 101.0 100.8 97.7 88.7 97.3 24100 101.4 104.9 98.4 103.8 106.0 95.6 84.2 97.8 103.1 25 100 103.8 108.4103.8 103.6 104.4 103.7 89.7 96.2 100.0 26 100 100.9 100.7 96.0 100.096.1 96.7 96.6 96.9 99.8 27 100 97.3 100.1 98.4 97.6 98.0 97.5 91.8 88.466.0 28 100 102.6 102.6 100.0 99.8 98.7 94.3 93.4 86.8 88.7

The addition of citrate or tartrate and zinc salts to the formulationsof oxytocin appeared beneficial for both the aerodynamic performance andoxytocin stability.

Example 3

A pregnant woman, 35 year old and in her second pregnancy has a historyof mild post-partum hemorrhaging in her first pregnancy is admitted tothe hospital at 40 weeks of pregnancy and in labor. Contractions aremonitor to occur 5 minutes apart by the attending obstetrician. Thepregnant woman is noted to be bleeding and delivers a healthy baby. Inthe operating room and immediately upon childbirth, the woman isadministered by oral inhalation a dry powder formulation containing asingle dose of 100 IU of oxytocin, 28% (w/w) citrate and 7% (w/w) zincchloride, in a single inhalation, using an inhalation system comprisinga high resistance inhaler as described in U.S. Pat. No. 8,484,129, whichdisclosure is incorporated herein by reference in its entirety for itsteaching of the relevant subject matter. The woman was kept in thehospital for 3 days and did not suffer any severe bleeding and wasreleased with her newborn baby.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification, and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects those of ordinary skill in the art toemploy such variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

Further, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed:
 1. A method of treating or preventing post-partumhemorrhaging comprising, administering to a subject in need of treatmenta dry powder pharmaceutical formulation comprising an oxytocin syntheticpeptide, a derivative or analog of said peptide; zinc citrate or zincchloride, and/or a pharmaceutically acceptable carrier or excipientwherein the formulation is stable for at least 40 weeks at 40° C., 75%relative humidity.
 2. The method of claim 1, wherein the formulationcomprises zinc chloride.
 3. The method of claim 1, wherein thepharmaceutically acceptable carrier or excipient is a sugar selectedfrom mannose, mannitol, trehalose, or sorbitol.
 4. The method of claim1, wherein the pharmaceutically acceptable carrier or excipient ispolyvinylpyrrolidone, polyethylene glycol, or a diketopiperazine.
 5. Themethod of claim 4, wherein the diketopiperazine is fumaryldiketopiperazine or succinyl diketopiperazine.
 6. The method of claim 1,wherein the zinc citrate or zinc chloride is in an amount ranging from100 to 20 equivalents per mole of the oxytocin, the oxytocin analog orderivative thereof.
 7. The method of claim 1, wherein said oxytocinsynthetic peptide, a derivative, or an analog of said peptide is in anamount comprising up to 200 IU.
 8. The method of claim 7, wherein saidoxytocin synthetic peptide, a derivative, or an analog of said peptideis in an amount comprising up to 150 IU.
 9. The method of claim 7,wherein said oxytocin synthetic peptide, a derivative, or an analog ofsaid peptide is in an amount comprising up to 100 IU.
 10. The method ofclaim 1, wherein said oxytocin synthetic peptide, a derivative, or ananalog of said peptide is in an amount comprising up to 200 IU.
 11. Themethod of claim 1, wherein said oxytocin synthetic peptide, aderivative, or an analog of said peptide is in an amount comprising upto 150 IU.
 12. The method of claim 1, wherein said oxytocin syntheticpeptide, a derivative, or an analog of said peptide is in an amountcomprising up to 100 IU.
 13. The method of claim 1, wherein saidoxytocin synthetic peptide, a derivative, or an analog of said peptideis in an amount comprising up to 50 IU, wherein the formulationcomprises zinc citrate.
 14. The method of claim 1, further comprising anamino acid selected from leucine, isoleucine, trileucine, cysteine,lysine, glycine, arginine, methionine, and histidine.
 15. The method ofclaim 14, wherein the amino acid is leucine.
 16. The method of claim 14,wherein the amino acid is isoleucine.
 17. The method of claim 14,wherein the amino acid is trileucine.
 18. The method of claim 14,wherein the amino acid is cysteine.
 19. The method of claim 14, whereinthe amino acid is lysine.
 20. The method of claim 14, wherein the aminoacid is glycine.
 21. The method of claim 14, wherein the amino acid isarginine.
 22. The method of claim 14, wherein the amino acid ismethionine.
 23. The method of claim 14, wherein the amino acid ishistidine.